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Topical glucocorticoids (GCs), such as hydrocortisone (HC), are the main drugs used to treat inflammatory skin conditions including eczema and psoriasis, but their longterm use is limited by the onset of side effects such as skin thinning, impairment of wound healing and systemic metabolic dysfunction. For this reason, there is a substantial need for new compounds with the same anti-inflammatory effects but fewer adverse effects. Previous studies have suggested 5α-tetrahydrocorticosterone (5α-THB) as a new, more selective anti-inflammatory compound; this steroid is a metabolite of the major endogenous GC in rodents, corticosterone (B). Preliminary data indicated that 5α-THB may be as effective as HC in reducing mouse irritant dermatitis, but without the local or systemic side effects of HC. The aim of this thesis is to investigate the mechanisms through which 5α-THB delivers more selective anti-inflammatory effects, with the hypothesis that 5α-THB influences distinct signalling pathways from those of B. A mouse model of irritant dermatitis induced by topical application of croton oil on the ear was developed, and the anti-inflammatory properties of 5α-THB were analysed, in comparison with those of B, after 6 and 24 hours of treatment. In inflamed tissue, B reduced tissue oedema and cell infiltration at both time points; in contrast, 5α-THB did so at 24 but not 6 hours, at a dose five-fold higher than B. Real-time analysis at 24 hours showed that B and 5α-THB similarly reduced the croton oil-induced increase of transcripts of genes encoding vascular and cellular adhesion molecules. Interestingly, while B did not affect the abundance of transcripts of the anti-inflammatory gene Dusp1, 5α-THB increased it in croton oil-treated ears, suggesting a different mechanism of action between 5α-THB and B. The experiment was repeated with the injection of the glucocorticoid receptor (GR) antagonist RU486; RU486 relieved the effect of B on swelling but did not attenuate the anti-inflammatory effects of 5α-THB, indicating a further important difference between the two steroids. Angiogenesis is fundamental for the healing process, and it is known that topical GCs impair wound healing in part by inhibiting angiogenesis; for this reason, the effects of 5α-THB on the formation of new vessels, in comparison with B, were tested in a mouse model of inflammatory angiogenesis induced by sub-cutaneous implantation of polyurethane sponges. 5α-THB, at equipotent doses to B for the reduction of macrophage infiltration, inhibited angiogenesis to a lesser extent than its precursor. In addition, B had systemic effects in that it lowered adrenal gland weights, whereas 5α-THB did not. Histological analysis suggested that while B inhibits formation and maturation of new vessels, 5α-THB may affect only the former process. Molecular analysis showed that B reduced the abundance of transcripts of the majority of the tested genes involved in inflammation, angiogenesis and tissue remodelling, but 5α-THB had more selective effects. Ex vivo studies in mouse bone marrow-derived macrophages stimulated with LPS showed that 5α-THB inhibited release of pro-inflammatory cytokines in a weaker manner compared with B. This inhibition was partially prevented by co-incubation of RU486 with B but not with 5α-THB. In in vitro studies, molecular pathways activated by B and associated with adverse side effects were only weakly activated by 5α-THB. In particular, 5α-THB only weakly induced phosphorylation of GR, and activation of expression of GC-responsive reporter plasmids and endogenous metabolic genes. Interestingly, 5α-THB reduced B-induced trans-activation of some of these genes. In summary, 5α-THB effectively reduces skin inflammation, but, unlike B, has only moderate anti-angiogenic properties, and weakly activates molecular mechanisms associated with adverse metabolic side effects. Most importantly, its action may not be due to activation of GR. This work opens the intriguing possibility that GCs work through mechanisms not yet investigated, and this may be of pivotal importance in the search for new safer anti-inflammatory compounds.

In patients with diabetes, hyperglycemia leads to functional impairment of endothelial cells (ECs) and microangiopathy. Inflammation and endothelial dysfunction (ED) have been associated with the development of several cardiovascular complications. Concentration of methylglyoxal (MG) - a highly reactive aldehyde is increased in diabetes. In a non-pathological state, MG is detoxified by the enzymes glyoxalase-1 (GLO1) and glyoxalase 2 in presence of glutathione. This thesis examines the role of MG accumulation in ECs and bone marrow cells (BMCs), with the consequences it has for their function. To this end, a transgenic mouse model was used in which the human enzyme GLO1 is overexpressed in the vasculature By using a GLO1 overexpressing mouse model studies described here examined the contribution of MG-induced inflammation in vivo to cardiovascular complications of diabetes, namely diabetic heart failure and peripheral vascular disease. This study confirmed that accumulation of MG leads to inflammation and cell death, and further explained how MG affects the role of ECs in development of the heart failure and BMCs in the revascularization. Overexpression of GLO1 in the vasculature diminished MG-induced inflammation, reduced EC death and delayed and limited the loss of cardiac function in streptozotocin (STZ)-induced diabetic mice (Chapter 2). The in vitro part of this study showed that MG and tumor necrosis factor (TNF- have a synergistic effect on cell death (Chapter 3). Overexpressing the GLO1 in BMCs only, restored neovascularization in ischaemic tissue of mice with STZ-induced diabetes (Chapter 4). Taken together, the results of this thesis suggest that hyperglycemia increased MG leads to endothelial inflammation, EC death and decreased angiogenic potential of BMCs. Furthermore, this MG-induced inflammation and reduced cell function observed, identifies a potential target for therapy of the cardiovascular complications seen in diabetes.

Cardiac performance, angiogenesis and growth was investigated during early chicken development. Heart rate, and thus arterial pulse pressure and cardiac output, were altered with the bradycardic drug ZD7288. Heart rates at 72 h of development of control embryos and those dosed with chicken Ringer were not different at 171 bpm. Acute and chronic application of ZD7288 caused significant bradycardia. Chronic dosing of Ringer and ZD7288 changed neither eye diameter nor development rate. Chronic dosing of ZD7288 did not significantly alter CAM vessel density close to the embryo (2, 3 and 4 mm) but at farther distances (5 and 6 mm) chronic dosing with both Ringer and ZD7288 decreased vessel density by 13 - 16%. Chronic dosing with ZD7288 also reduced body mass by 20%. Thus, lowered heart rate and cardiac output had little effect on vessel density or developmental stage, but did reduce embryo growth.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17781号 / 医博第3807号 / 新制||医||999(附属図書館) / 30588 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 上本 伸二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Our previous studies indicate that 3, 4-(methylenedioxy)-1-(2′, 3′-epoxypropyl)-benzene (safrole oxide), a newly synthesized compound, induces apoptosis in vascular endothelial cells (VECs) and A549 lung cancer cells. To our knowledge, the inhibition of angiogenesis by safrole oxide has not been reported yet. We report here that cultured rat aorta treated with safrole oxide exhibited a significant microvessel reduction as determined by counting the number of microvessels in a phase contrast microscope. There were more microvessels formed in the presence of A549 lung cancer cells in rat aorta model, while a dramatic inhibition of angiogenesis was obtained by adding 220-450 μmol l- 1 of safrole oxide to the growth medium (P <. 01). The culture of rat aorta treated with safrole oxide produced only some abortive endothelial cells but not microvessels. Furthermore, safrole oxide induced antiangiogenic effect in the chorioallantoic membranes (CAM) as a dose dependent manner. Eggs treated with 2-11 μmol 100 μl- 1 per egg of the safrole oxide for 48 h exhibited a significant reduction in blood vessel area of the CAM, a process likely mediated by apoptosis as demonstrated by DNA fragmentation. Our results suggest that safrole oxide has antiangiogenic activity and this effect might occur by induction of cellular apoptosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Abnormal blood vessel growth from pre-existing blood vessels, termed pathological angiogenesis, is a common characteristic of vascular diseases like proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (wet AMD). Retinal detachment, hemorrhage, and loss of vision are only some of the debilitating consequences of advanced pathological angiogenesis. Current therapeutics targeting these blood vessels are ineffective in many patients. We previously identified a novel angiogenesis target, ferrochelatase (FECH), from the heme synthesis pathway, and found that FECH inhibition is antiangiogenic in cell and animal models. Heme synthesis occurs in mitochondria, where FECH inserts Fe2+ into protoporphyrin IX (PPIX) to produce heme. However, the relationship between heme metabolism and angiogenesis is poorly understood. I sought to understand the mechanism of how FECH and thus, heme is involved in endothelial cell function. First, I determined the energetic state of retinal and choroidal endothelial cells, previously uncharacterized. I found that mitochondria in endothelial cells had several functional defects after heme inhibition. FECH loss changed the shape of mitochondria and depleted expression of genes maintaining mitochondrial dynamics. FECH blockade elevated oxidative stress and depolarized mitochondrial membrane potential. Heme depletion had negative effects on cellular metabolism, affecting oxidative phosphorylation and glycolysis. Mitochondrial complex IV of the electron transport chain (cytochrome c oxidase) was decreased in cultured human retinal endothelial cells and in murine retina ex vivo after FECH inhibition. Supplementation with heme partially rescued phenotypes of FECH blockade. Additionally, I discovered that partial loss-of-function Fech mutation in mice caused PPIX accumulation with no change in normal vasculature, as assessed by fundoscopy. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. My studies identify a novel role of a heme synthesis enzyme in blood vessel formation and provide an opportunity to exploit these findings therapeutically for patients with PDR and wet AMD.

Solid tumors typically require angiogenesis, the development of new blood vessels, for growth and metastasis. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating receptors on host endothelial cells. One such receptor, Flt-1, occurs as either a membrane bound or a secreted form (sFlt-1) that can inhibit angiogenic signaling. Previous studies have shown that variation in mRNA expression of VEGF and its receptors KDR, sFlt-1 and Flt-1 occurs in pathological angiogenesis, i.e. metastatic tumorigenesis. We hypothesize that the ratio of sFlt-1:Flt-1 mRNA will be altered in the presence of solid tumors. The objective of this study was to evaluate the expression of sFlt-1 and Flt-1 mRNAs in a mouse metastatic tumor model using CT26.CL25 cells. CT26.CL25 cells are VEGF-producing murine colon carcinoma cells transfected with the lacZ gene, which expresses B-galactosidase activity. These cells, injected intravenously, form tumor nodules in the lung. A pilot study revealed development of lung nodules in mice nine days after intravenous injection with 105 cells. In a second study, twenty-five 10- week-old female Balb/c mice were injected intravenously, via tail vein, with 2 x 105cells, and fifteen with vehicle control. Lung nodules developed in all mice injected with cells. Tissues were harvested by routine necropsy and either formalin-fixed for routine histology/histochemistry or stored for quantitative RT-PCR (QPCR) analysis of gene expression. Under microscopic evaluation, sections of lungs stained with Hematoxylin & Eosin (H&E) revealed nodules composed of polygonal neoplastic cells. cDNA from lungs (14 tumor-bearing, 10 controls) and cultured CT26.CL25 cells was analyzed by QPCR using primers and TaqMan probes directed against sFlt-1, Flt-1, KDR, VEGFA, PlGF (Placental Growth Factor), Angiotensin Converting Enzyme (ACE), 18S ribosomal RNA and neoR (neomycin phosphotransferase). We observed an increased sFlt-1:Flt-1 ratio in tumor-bearing versus control lungs, suggesting that tumor-derived signals may influence sFlt-1 and Flt-1 expression differentially. Additionally, there was increased expression of Flt-1, sFlt-1 and KDR in tumors versus controls, but not in VEGF expression in tumors versus controls. Interestingly, expression of PlGF was increased in tumors versus controls, suggesting its role as an enhancer of tumor progression in the presence of other angiogenic factors. Together, these findings indicate that solid tumor angiogenesis results from an intricate balance of various angiogenic factors. / Master of Science

Melanoma is a skin cancer that relies on angiogenesis for growth and progression. Angiogenesis is the growth of new vessels from existing vessels and follows a number of steps that include endothelial cell growth, migration and tubulogenesis. Current anti-angiogenic drugs are not effective in the treatment of melanomas due to serious side effects such as hypertension and the development of resistance. On the other hand, gold nanoparticles (AuNPs) have been reported to be biocompatible in preclinical models. Furthermore, AuNPs were shown to be cytotoxic to prostate cancer cells. In human umbilical vein endothelial cells, AuNPs inhibited the angiogenic protein, vascular endothelial growth factor-A (VEGF-A). Therefore, the study aimed to investigate the possible cytotoxic effects of AuNPs (1.2–3.2 nM) on melanoma cells and angiogenesis parameters (endothelial cell growth and migration) as well as on the levels of angiogenesis promoting proteins, VEGF-A and placental growth factor (PIGF). Melanoma (B16-F10) cells and tumour-derived endothelial (sEnd.2) cells were maintained in an incubator in a humidified atmosphere containing 5% CO2 at a temperature of 37°C. To investigate whether AuNPs were cytotoxic to melanoma cells, the effect of the particles on B16-F10 cell survival was measured using the crystal violet assay. To determine the effects of AuNPs on angiogenesis parameters, endothelial cell (EC) growth and migration were investigated using crystal violet assay and the scratch assay respectively. Also, EC morphology was studied using polarisation-optical interference contrast light microscopy. The enzyme-linked immunosorbent assay (ELISA) was used to determine the effects of AuNPs on the levels of VEGF-A and PIGF. The results showed that AuNPs decreased the viability of melanoma and endothelial cells. The scratch assay showed that more ECs migrated in cultured treated with AuNPs (P < 0.05). The concentration of VEGF-A and PIGF was reduced significantly following treatment with AuNPs, meaning that the particles exhibited anti-angiogenic properties. This outcome provides a basis for further testing of AuNPs as a potential treatment for melanoma. / Dissertation (MSc)--University of Pretoria, 2020. / National Research Foundation / Mintek / Physiology / MSc / Unrestricted